Methods to enhance delivery and engraftment of stem cells including the identification of specific prostagandin e2 receptors

ABSTRACT

The receptor EP4 is identified as the PGE2 receptor that is most responsible enhancing the homing and engraftment of hematopoietic stem and progenitor cells. Treatment of graft sources and graft recipients with compounds that preferentially target the EP4 receptor provide effective methods of increasing engraftment success while minimizing adverse side effects that may Obe associated with therapies that include the use of less selective molecules such as PGE2 and dmPGE2. One effective molecule used in such therapies is 5-[(1E,3R)-4,4-ditluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof (L-902, 685).

PRIORITY CLAIM

This application claims the benefit of U.S. provisional patent application Ser. No. 61/261,349 filed on Nov. 15, 2009 and of U.S. provisional patent application No. 61/261,352 filed on Nov. 15, 2009, each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL RIGHTS

Part of the work during the development of this invention was made with government support from the National Institute of Health (NIH) under grant number HL069669. The U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

Methods of treating potential cell grafts comprising hematopoietic stem and progenitor cells with a selective receptor agonist of the EP4 receptor to enhance their homing, survival, self-renewal and proliferation.

BACKGROUND

Bone marrow transplantation, including the more popular procedures of mobilized peripheral blood stem cell transplantation and umbilical cord blood transplantation are routinely used as curative procedures for malignant and nonmalignant hematologic diseases and genetic disorders. These procedures require that hematopoietic grafts containing sufficient numbers of stems and progenitor cell populations be harvested from healthy normal donors or from patients at a time of low or absent disease and subsequently administered to patients whose hematopoietic system and presumably disease tissue has been eradicated. After transplantation, the appropriate stem cells travel to or “home” to the appropriate bone marrow micro-environmental niches. Once lodged within the appropriate niches, these cells proliferate and produce new stem cells, a process called self-renewal. The cells also differentiate into lineage restricted progenitor cells and mature cells, thereby restoring the blood forming hematopoietic system for the life of the recipient. Progenitor cells are required in said grafts to also produce mature cells; however since they are not stem cells and cannot self-renew, their participation is limited in lifespan. Successful transplantation procedures require that sufficient cells be collected from the donor and administered to the recipient. The need for large numbers of cells is aggravated by the fact that collection procedures and the process of homing and engraftment are stressful to the graft cells resulting in the loss of a portion of the cells in the graft.

In particular, umbilical cord blood grafts contain limited numbers of stem cells and for this reason usually cannot be routinely used to transplant adults. Similarly, 10-25% of normal donors and up to 75% of specific patient populations, particularly those exposed to certain chemotherapeutic agents, e.g., fludarabine, fail to mobilize sufficient cells for use in transplant procedures. In general, the greater the number of viable cells that can be transplanted the greater the chances are for a successful treatment. Accordingly, there is a need for novel agents and/or methodologies that can increase the number of hematopoietic stem cells or progenitor cells in the transplant or alternatively to facilitate or enhance their homing to bone marrow. Some aspects of the current invention seek to address this need.

SUMMARY

Some aspects of the present invention provided methods of treating a donor or donor cells or a recipient of hematopoietic stem or progenitor cells, comprising the steps of providing at least one compound that preferentially interacts with the PGE₂ EP₄ receptor, on hematopoietic stem or progenitor cells, or a pharmaceutically acceptable salt thereof and administering a therapeutically acceptable dose of said compound to a patient in need thereof. These compounds may be selected from the group consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE; 5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid)

In some embodiments the compound is 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yphexyl]-2-pyrrolidinone (L-902,688). In some embodiments, the patient is recipient of a graft, wherein the graft includes at least one type of cell selected from the group consisting of hematopoietic stem cells and progenitor cells that have treated with an agonist that preferentially binds to the EP₄ receptor.

Some embodiments include methods of treating a donor, donor cells or a recipient of hematopoietic stem or progenitor cells, comprising the steps of: providing at least one compound that preferentially interacts with the PGE₂ EP₄ receptor, on hematopoietic stem or progenitor cells, or a pharmaceutically acceptable salt thereof; and administering a therapeutically acceptable dose of said compound to a patient in which the patient is a donor or a recipient of hematopoietic stem or progenitor cells. In many of these methods the compound increases the homing and/or the engraftment of the hematopoietic stem or progenitor cells.

Compounds that interact with the PGE₂ EP₄ receptor include, but are not limited to, compounds selected from the groups consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE; 5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3 -(trifluoromethyl)phenyl]butyl}-5 -oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-{(1R,4S,5 R)-5-[(E)-5-(3 -chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) or a pharmaceutically acceptable salt thereof. Still another compound that interacts with PGE₂ EP₄ receptor and can be used in some embodiments of the invention is the compound 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof.

In some embodiments recipient graft includes at least one type of cell selected from the group consisting of hematopoietic stem cells and progenitor cells that are treated with an EP₄ agonist. Either human or animal patients may be treated with these compounds or with cells that were first treated with these compounds either in vivo or in vitro.

Still other embodiments of the invention include methods of treating a human or an animal patient, comprising the steps of: providing a therapeutically effective amount of a PGE₂ EP₄ agonist or a pharmaceutically acceptable salt thereof; harvesting a hematopoietic stem or progenitor cell from a donor; and contacting said PGE₂ EP₄ agonist to a hematopoietic stem or progenitor cell, wherein said hematopoietic stem or progenitor cell was harvested from the donor.

Compounds that can be contacted with hematopoietic stem or progenitor cells in order to practice the invention include, but are not limited to, compounds selected from the group consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3 S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE; 5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) or a pharmaceutically acceptable salt thereof. Still another compound that can be used to practice the invention is 5 -[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof. In some embodiments, hematopoietic stem or progenitor cells treated with these compounds or other PGE₂ EP₄ agonist are then administered to a human or an animal patient.

In some embodiments the therapeutically effective amount of the PGE₂ EP₄ agonist contacted with the hematopoietic stem or progenitor cells is on the order of between about 0.001 μM to about 10 μM per about 1.0×10⁶ cell per ml to about 1.0×10⁷ cells per ml of said hematopoietic stem or progenitor cells. In some embodiments the hematopoietic stem or progenitor cells treated and used to treat a human or animal recipient are harvested from marrow, umbilical cord or peripheral blood obtained from a human or an animal donor. In some embodiments the donor and the recipient of the hematopoietic stem or progenitor cells are the same human or animal patient.

Some embodiments of the invention include a method for altering the activity of a cell, comprising the steps of: providing a hematopoietic stem or progenitor cell, wherein the cells express at least one PGE₂ EP₄ receptor; supplying at least one compound that preferentially interacts with the PGE₂ EP₄ receptor or a pharmaceutically acceptable salt thereof; and contacting the hematopoietic stem or progenitor cell with said compound. In some embodiments the hematopoietic stem or progenitor cell are isolated from marrow, umbilical cord or peripheral blood. In some embodiments contacting the cells with the compound(s) increases the homing of said cells and or the engrafting potential of the cells. In some embodiments the compounds contacted with the cells includes, but is not limited to, at least one compound selected from the group consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE; 5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenylibutyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) or a pharmaceutically acceptable salt thereof. In some embodiments at least one of the compound is 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof. In some embodiments the amount of therapeutically effective compound contacting said cells is about 0.001-10 microMolar. In some embodiments the number of cells treated with the compound is on the order of between about 10⁶ to about 10⁷ cells per mL.

Other aspects of the present invention provide methods of treating a donor, donor cells or a recipient of hematopoietic stem or progenitor cells comprising the step of administering to the donor, donor cells or recipient a therapeutically effective amount of an EP₄ agonist.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Graph illustrating that treatment of cells with EP₄ agonist ex vivo specifically up regulates CXCR4 expression on CD34⁺ cells.

FIG. 2 . Graph illustrating that signaling via the EP₄ receptor is responsible for up regulation of CXCR4 expression.

DESCRIPTION

For the purposes of promoting an understanding of the principles of the novel technology, reference will now be made to the preferred embodiments thereof, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the novel technology is thereby intended, such alterations, modifications, and further applications of the principles of the novel technology being contemplated as would normally occur to one skilled in the art to which the novel technology relates.

Unless stated otherwise the term, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound that when administered to a human being or an animal patient or to a cell or collection of cells either alone or in combination with other pharmaceutically active ingredients or other components of medicaments that have a desirable effect on the physiological condition of a patient or the cell or collection of cells.

Therapeutically effective, beneficial or efficacious doses of various compounds that preferentially bind to PGE₂ EP₄ receptors administered in vivo to either a human or an animal patient are in the range of between about 0.1 mg of the compound per Kg of body weight of the patient per day to about 100 mg of the compound per Kg of body weight of the patient per day.

Compounds that preferentially bind to PGE₂ EP₄ receptors are compounds that have a higher affinity for the EP₄ receptor than for any of the other three EP receptors namely EP₁, EP₁ and EP₃.

Compounds that can be used to practice some embodiments of the invention include, but are not limited to, the following: 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-16-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone also referred to as L-902,688 (Young, et al., 2004); 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid also referred to as ONO-AE1-329 (Suzawa et al., 2000); methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate also referred to as ONO-4819 (Maruyama et al., 2002; Ohta et al., 2009); 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE₁ (Maruyama et al., 2002); 5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl{-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate also referred to as PF-04475270 (Luu et al., 2009); APS-999 Na (El-Nefiawy et al., 2005); [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethy1]biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide] (Machwate et al., 2001); and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) U. S. Patent application number US2005/0164992A1, Jul. 28, 2005, to Donde Y, Nguyen J H, Kedzie K M, Gil D M, Donello J E and Im W B.

Unless stated otherwise the term “about” as used herein refers to range of value of plus or minus 10%, e.g., ‘about 1.0’ includes values between 0.9 and 1.1.

Treatment of bone marrow cells, umbilical cord blood cells, mobilized peripheral blood cells or any hematopoietic cell graft to be used for hematopoietic transplantation with Prostaglandin E₂ (PGE₂) or any active analogue or metabolite of PGE₂ or any E series prostaglandin with specificity for the PGE₂ EP₄ receptor, improves the homing, survival and proliferation of the transplanted hematopoietic stem cells. This treatment can be used to increase stem cell engraftment rates and thereby improve the efficiency of hematopoietic stem cell transplantation.

By some estimates the success of peripheral blood stem cell transplantation requires administration of approximately 2 million CD34⁺ cells per kilogram of recipient patient body weight. Any agent, combination of agents or manipulations that increases the number of stem cells that can be collected, enhances their survival rates, enhances their ability to home to the appropriate marrow environment and/or enhances their self-renewal and proliferation rates will likely have a positive impact on the efficacy of hematopoietic transplantation procedures. The success of these procedures may be measured in terms of reduced patient morbidity and mortality. Numerous studies have been undertaken to try and expand the number of human hematopoietic stem cells within isolated grafts in ex vivo settings, with limited success (Broxmeyer, 2006; Haylock and Nilsson, 2007). Recently, the CXCR4 antagonist AMD3100 has been shown to enhance mobilization of stem cells (Broxmeyer, et al., 2005; Liles, et al., 2003) and in clinical trials, (Plerixafor; Mozibil) has been shown to enhance collection of mobilized stem cells when used in combination with G-CSF (DiPersio et al., 2007b; DiPersio et al., 2007a). Truncation of chemokines has been used as a method to enhance the body's ability to mobilize stem cells. Some of these methods have been patented, e.g., U.S. Pat. Nos. 6,080,398; 6,447,766B1; 639,053B1; 6,713,052, each of which is incorporated by reference in its entirety. Their ability to more efficiently mobilize stem cells has also been reported (King, et al., 2001; Pelus, et al., 2004). A role for blocking the activity of a surface peptidase (CD26) has been reported as a method for enhancing the homing of hematopoietic stem cells (Christopherson, et al., 2004).

A number of agents when used in combination with G-CSF have been reported to increase the number of hematopoietic progenitor cells that can be recovered (Pelus and Fukuda, 2007; Herbert, et al., 2007), however, the ability of these agents to mobilize the long-term repopulating stem cells, i.e., the stem cells with self-renewal activity, has not been uniformly demonstrated. A recent study has shown that pulse exposure of mouse bone marrow cells to 16,16 dimethyl pGE₂ (dmpGE₂) enhances engraftment of hematopoietic stem cells, however this study provides no evidence of mechanism of action and specifically states that the effect of PGE₂ is not on cell homing (North, et al., 2007). It was unexpectedly demonstrated by Hoggatt, et al., 2009, that PGE₂ increases the CXCR4 receptor on hematopoietic stem and progenitor cells, and that this increase is responsible for increasing the homing to the bone marrow niche, resulting in a subsequent increase in engraftment.

It is generally believed that PGE₂ interacts with 4 highly conserved G-protein coupled receptors (GPCR); EP₁, EP₂, EP₃, and EP₄ that account for the multiple, sometimes opposing effects attributed to PGE₂ (Breyer, et al., 2001). EP receptor expression levels vary among different tissues, with EP₃ and EP₄ mRNA being most abundant (Sugimoto and Narumiya, 2007a) and EP₂ mRNA expressed at lower levels than EP₄ in most tissues (Katsuyama, et al., 1995). EP₁ activates phospholipase C (PLC) via an unidentified G protein (Tsuboi, et al., 2002), which increases intracellular Ca²⁺ coupled to inositol phosphates resulting in activation of phosphokinase C (PKC) (Breyer, et al., 2001). EP₃ receptor ligation results in inhibition of adenylate cyclase and decreased cAMP that is Gα_(i) linked (Sugimoto, et al., 2007). Multiple EP₃ splice variants have been identified and depending on C-terminal splicing, they can couple to multiple G proteins (Namba, et al., 1993). EP₂ and EP₄ both couple to G_(α)s leading to adenylate cyclase activation and increased cAMP, activating protein kinase A (PKA), as well as Rap1, Rac1, and PKCζ (PKC zeta), a unique isoform implicated in HSC function (Goichberg, et al., 2006). EP₂ and EP₄ are thought to have partially redundant roles in some systems, while in others they play distinct roles (Sugimoto and Narumiya, 2007). EP₄ but not EP₂ can activate the PI3K/Akt pathway in addition to adenylate cyclase (Fujino, et al., 2003). EP₄ has a longer cytosolic domain allowing for more ligand dependent phosphorylation and more rapid desensitization (Nishigaki, et al., 1996) enabling a selective negative feedback loop (Sugimoto and Narumiya, 2007). Lastly, EP₄ is internalized when activated, while EP₂ is not (Desai, et al., 2000). As a consequence, EP₂ and EP₄ can have different roles based upon continuation or attenuation of signals generated by receptor activation (Breyer, et al., 2001). Treating with PGE₂ often exhibits a “bell-shaped” dose-response curve suggesting a different repertoire of EP receptors is activated dependent upon PGE₂ concentration (Hull, et al., 2004).

Most current strategies to improve hematopoietic transplantation utilizing prostaglandin have used either native PGE₂ or a long acting derivative of PGE₂, 16,16 dimethylprostaglandin E₂ (dmPGE₂). These prostaglandin compounds are thought to activate all 4 EP receptors leading to the numerous downstream signaling events briefly described above. As demonstrated herein, the enhancement in homing and engraftment of hematopoietic stem and progenitor cells is due to up regulation of the CXCR4 receptor by treatment with prostaglandin. Specifically focusing on the EP receptor that is responsible for the increase in CXCR4, (the EP₄ receptor) has the benefit of enhancing the grafting process without activating receptors that may be detrimental to the engraftment process and/or have other unknown possible deleterious consequences.

Treating with an E4 Selective Agonist Affects Homing and Engraftment Efficiency

Un-manipulated hematopoietic grafts or purified hematopoietic stem cell populations (e.g., SKL cells in mice or CD34 cells in humans) are incubated with an EP₄ specific agonist: 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone (i.e. L-902,688), on ice or at room temperature at concentrations of 0.001-10 microMolar agonist per 1-10 million cells in 1 ml of culture medium, e.g. IMDM, for 15 minutes-6 hrs. After incubation, the cells are washed 3 times in sterile media or saline and administered to recipients, intravenously. L-902,688 was a generous gift from Merck Frosst (Kirkland, Canada) (Young, et al., 2004).

Referring now to FIG. 1 . This graph illustrates that treatment of cells with EP₄ agonist ex vivo specifically up regulates CXCR4 expression on CD34⁺ cells. The insert shows cytometry histograms of CD34⁺ cells from cord blood samples showing significant up regulation of CXCR4 on the surface of CD34⁺ cells after pulse exposure to EP₄ agonist (light line) and dmPGE₂ (dark line) compared to isotype control (grey shaded area). The bar graph indicator data measured from such experiments graphed as a function of different compounds used dmPGE₂, Butaprostone, sulprostone and EP₄.

Referring now to FIG. 2 . This graph illustrates that signaling via the EP₄ receptor is responsible for up regulation of CXCR4 expression. The insert shows data for 3 cord blood samples. Treatment with EP₄ agonist ex vivo up regulates CXCR4 expression on CD34^(|) cells. The bar chart shows combined data for 8 samples. The bar chart data demonstrates that ex vivo treatment with EP₄ agonist up regulates CXCR4 by about 3 fold. In the bar chart this effort is normalized to 100%. Pretreatment of cells with a specific antagonist of the EP 1, 2 and 3 receptor prior to exposure to EP₄ antagonist had no effect on up regulation of CXCR4 whereas pre-incubation with a selective EP₄ antagonist prior to exposure to EP₄ agonist significantly blocked up regulation of CXCR4. The fact that up regulation of CXCR4 by an EP₄ selective agonist is blocked by a selective EP₄ antagonist, but not by EP1, 2, 3, antagonist, provides additional evidence that up regulation is mediated through the EP₄ receptor.

Additional embodiments include administering EP₄ agonists (e.g., on the order of about 0.001-10 microMolar) to patients immediately prior to and daily afterwards after receiving a hematopoietic graft as a means of enhancing stem cell function. Therapeutic effective doses are amounts of the pharmaceutically active agent used either alone or in combination with another pharmaceutical agent or inert material that has a beneficial effect on a so treated human or animal patient. Such benefits may include, but arc not limited to, increasing the effectiveness of other steps in a given treatment regimen.

While the novel technology has been illustrated and described in detail in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the novel technology are desired to be protected. As well, while the novel technology was illustrated using specific examples, theoretical arguments, accounts, and illustrations, these illustrations and the accompanying discussion should by no means be interpreted as limiting the technology. All patents, patent applications, and references to texts, scientific treatises, publications, and the like referenced in this application are incorporated herein by reference in their entirety.

REFERENCES

-   Breyer, R. M., Bagdassarian, C. K., Myers, S. A., and Breyer, M. D.     (2001). Prostanoid receptors: subtypes and signaling. Annu. Rev.     Pharmacol. Toxicol. 41, 661-690. -   Broxmeyer, H. E. (2006). Cord Blood Hematopoietic Stem and     Progenitor Cells. In Essentials of Stem Cell Biology, Elsevier,     Inc.), pp. 133-137. -   Broxmeyer, H. E., Orschell, C. M., Clapp, D. W., Hangoc, G., Cooper,     S., Plett, P. A., Liles, W. C., Li, X., Graham-Evans, B.,     Campbell, T. B., Calandra, G., Bridger, G., Dale, D. C., and     Srour, E. F. (2005). Rapid mobilization of murine and human     hematopoietic stem and progenitor cells with AMD3100, a CXCR4     antagonist. J. Exp. Med. 201, 1307-1318. -   Christopherson, K. W., Hangoc, G., Mantel, C. R., and     Broxmeyer, H. E. (2004). Modulation of hematopoietic stem cell     homing and engraftment by CD26. Science 305, 1000-1003. -   Desai, S., April, H., Nwaneshiudu, C., and Ashby, B. (2000).     Comparison of agonist-induced internalization of the human EP2 and     EP4 prostaglandin receptors: role of the carboxyl terminus in EP4     receptor sequestration. Mol. Pharmacol. 58, 1279-1286. -   DiPersio, J. F., Micallef, I., Stiff, P., Bolwell, B. J.,     Maziarz, R. T., Angell, J., Bridger, G., and Calandra, G. A phase     III, multicenter, randomized, double bnlind, placebo-controlled,     comparative trial of AMD3100 (Perixafor)+G-CSF vs. G-CSF+placebo for     mobilization in Non-Hodgkins Lymphoma (NHL) patients for autologous     hematopoietic stem cell (aHSC) transplantation. Blood 110, 185a.     2007a. -   Ref Type: Abstract -   DiPersio, J. F., Stadtmauer, A. P., Nademanee, A. P., Stiff, P.,     Micallef, I., Angell, J., Bridger, G., and Calandra, G. A phase III,     multicenter, randomized, double bnlind, placebo-controlled,     comparative trial of AMD3100 (Perixafor)+G-CSF vs. G-CSF+placebo for     mobilization in multiple myeloma (MM) patients for autologous     hematopoietic stem cell (aHSC) transplantation. Blood 110, 137a.     2007b. -   Ref Type: Abstract -   El-Nefiawy, N., Abdel-Hakim, K., and Kanayama, N. (2005). The     selective prostaglandin EP4 agonist, APS-999 Na, induces follicular     growth and maturation in the rat ovary. Eur. J. Endocrinol. 152,     315-323. -   Fujino H, Xu W, Regan J W. Prostaglandin E2 induced functional     expression of early growth response factor-1 by EP4, but not EP2,     prostanoid receptors via the phosphatidylinositol 3-kinase and     extracellular signal-regulated kinases. J. Biol. Chem. 2003;     278:12151-12156. -   Goichberg P, Kalinkovich A, Borodovsky N et al. cAMP-induced PKCzeta     activation increases functional CXCR4 expression on human CD34+     hematopoietic progenitors. Blood 2006; 107:870-879. -   Haylock, D. N. and Nilsson, S. K. (2007). Expansion of umbilical     cord blood for clinical transplantation. Curr Stem Cell Res Ther 2,     324-335. -   Herbert, K. E., Walkley, C. R., Winkler, I. G., Hendy, J., Olsen, G.     H., Yuan, Y. D., Chandraratna, R. A., Prince, H. M., Levesque, J.     P., and Purton, L. E. (2007). Granulocyte colony-stimulating factor     and an RARalpha specific agonist, VTP195183, synergize to enhance     the mobilization of hematopoietic progenitor cells. Transplantation     83, 375-384. -   Hull, M. A., Ko, S. C., and Hawcroft, G. (2004). Prostaglandin EP     receptors: targets for treatment and prevention of colorectal     cancer? Mol. Cancer Ther. 3, 1031-1039. -   Katsuyama, M., Nishigaki, N., Sugimoto, Y., Morimoto, K., Negishi,     M., Narumiya, S., and Ichikawa, A. (1995). The mouse prostaglandin E     receptor EP2 subtype: cloning, expression, and northern blot     analysis. FEBS Lctt. 372, 151-156. -   King, A. G., Horowitz, D., Dillon, S. B., Levin, R., Farese, A. M.,     MacVittie, T. J., and Pelus, L. M. (2001). Rapid mobilization of     murine hematopoietic stem cells with enhanced engraftment properties     and evaluation of hematopoietic progenitor cell mobilization in     rhesus monkeys by a single injection of SB-251353, a specific     truncated form of the human CXC chemokine GRObeta. Blood 97,     1534-1542. -   Liles, W. C., Broxmeyer, H. E., Rodger, E., Wood, B., Hubel, K.,     Cooper, S., Hangoc, G., Bridger, G. J., Henson, G. W., Calandra, G.,     and Dale, D. C. (2003). Mobilization of hematopoietic progenitor     cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood     102, 2728-2730. -   Luu, K. T. , Zhang, E. Y., Prasanna, G., Xiang, C., Anderson, S.,     Fortncr, J., and Vicini, P. (2009). Pharmacokinetic-pharmacodynamic     and response sensitization modeling of the intraocular     pressure-lowering effect of the EP4 Agonist     5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrr     olidin-1-yl]propyl}thiophene-2-carboxyl ate (PF-04475270). J.     Pharmacol. Exp Ther. 331, 627-635. -   Machwate, M., Harada, S., Leu, C. T., Seedor, G., Gallant, M.,     Hutchins, S., Lachance, N., Sawyer, N., Slipetz, D., Metters, K. M.,     Rodan, S. B., Young, R., and Rodan, G. A. (2001). Prostaglandin     receptor EP(4) mediates the bone anabolic effects of PGE(2). Mol.     Pharmacol. 60, 36-41. -   Maruyama, T., Kuwabe, S. I., Kawanaka, Y., Shiraishi, T., Shinagawa,     Y., Sakata, K., Seki, A., Kishida, Y., Yoshida, H., Maruyama, T.,     Ohuchida, S., Nakai, H., Hashimoto, S., Kawamura, M., Kondo, K., and     Toda, M. (2002). Design and synthesis of a selective EP4-receptor     agonist. Part 4: practical synthesis and biological evaluation of a     novel highly selective EP4-receptor agonist. Bioorg. Med. Chem. 10,     2103-2110. -   Namba T, Sugimoto Y, Negishi M et al. Alternative splicing of     C-terminal tail of prostaglandin E receptor subtype EP3 determines     G-protein specificity. Nature 1993; 365:166-170. -   Nishigaki, N., Negishi, M., and Ichikawa,A. (1996). Two Gs-coupled     prostaglandin E receptor subtypes, EP2 and EP4, differ in     desensitization and sensitivity to the metabolic inactivation of the     agonist. Mol. Pharmacol. 50, 1031-1037. -   North, T. E., Goessling, W., Walkley, C. R., Lengerke, C.,     Kopani, K. R., Lord, A. M., Weber, G. J., Bowman, T. V., Jang, I.     H., Grosser, T., Fitzgerald, G. A., Daley, G. Q., Orkin, S. H., and     Zon, L. I. (2007). Prostaglandin E2 regulates vertebrate     haematopoietic stem cell homeostasis. Nature 447, 1007-1011. -   Ohta, C., Kuwabe, S. I., Shiraishi, T., Shinohara, I., Araki, H.,     Sakuyama, S., Makihara, T., Kawanaka, Y., Ohuchida, S., and Seko, T.     (2009). An improved synthesis of the selective EP4 receptor agonist     ONO-4819. J. Org. Chem. 74, 8298-8308. -   Pelus, L. M., Bian, H., King, A. G., and Fukuda,S. (2004).     Neutrophil-derived MMP-9 mediates synergistic mobilization of     hematopoietic stem and progenitor cells by the combination of GCSF     and the chemokines GRObeta/CXCL2 and GRObetaT/CXCL2delta4. Blood     103, 110-119. -   Pelus,L. M. and Fukuda,S. (2007). Chemokine-mobilized adult stem     cells; defining a better hematopoietic graft. Leukemia. -   Sugimoto, Y. and Narumiya, S. (2007a). Prostaglandin E receptors. J.     Biol. Chem. 282, 11613-11617. -   Suzawa, T., Miyaura, C., Inada, M., Maruyama, T., Sugimoto, Y.,     Ushikubi, F., Ichikawa, A., Narumiya, S., and Suda, T. (2000). The     role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4)     in bone resorption: an analysis using specific agonists for the     respective EPs. Endocrinology 141, 1554-1559. -   Tsuboi, K., Sugimoto, Y., and Ichikawa, A. (2002). Prostanoid     receptor subtypes. Prostaglandins Other Lipid Mediat. 68-69,     535-556. -   Young, R. N., Billot, X., Han, Y., Slipetz, D. A., Chauret, N,     Belley, M., Metters, K., Mathieu, M, C., Greig, G. M., Denis, D.,     (2004), Discovery and synthesis of a potent, selective and orally     bioavailable EP4 receptor agonist. Heterocycles 64: 437-466. 

1. A method of treating a donor, donor cells or a recipient of hematopoietic stem or progenitor cells, comprising the steps of: providing at least one compound that preferentially interacts with a PGE2 EP4 receptor on a hematopoietic stem or progenitor cell, or a pharmaceutically acceptable salt thereof; and administering a therapeutically acceptable dose of said at least one compound to a patient where the patient is a donor or a recipient of the hematopoietic stem or progenitor cells.
 2. The method according to claim 1, wherein the compound increases the homing and/or engraftment of the hematopoietic stem or progenitor cells.
 3. The method according to claim 1, wherein said at least one compound is selected from the groups consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5 -[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethyl sulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE; 5-{3-[(2S)-2-(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}hept-5-enoic acid) or a pharmaceutically acceptable salt thereof.
 4. The method according to claim 1, wherein said at least one compound is 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-146-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof.
 5. The method according to claim 1, wherein the patient is the recipient of a graft wherein the graft includes at least one type of cell selected from the group consisting of hematopoietic stem cells and progenitor cells, wherein the cells have been treated with said at least one compound that preferentially binds to the PGE2 EP4 receptor.
 6. The method according to claim 1, wherein the patient is a human.
 7. The method according to claim 1, wherein the patient is an animal.
 8. A method of treating a patient, comprising the steps of: providing a therapeutically effective amount of a PGE2 EP4 agonist or a pharmaceutically acceptable salt thereof; and harvesting a hematopoietic stem or progenitor cell from a donor; and contacting said PGE2 EP4 agonist with a hematopoietic stem or progenitor cell, wherein said hematopoietic stem or progenitor cell was harvested from the donor.
 9. The method according to claim 8, wherein said at least one compound is selected from the groups consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethyl sulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE; 5-{3-[(2S)-2-(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3 -butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) or a pharmaceutically acceptable salt thereof.
 10. The method according to claim 8, wherein said at least one compound is 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof.
 11. The method according to claim 8, further comprising the step of: administering the hematopoietic stem or progenitor cell to a patient, wherein said cells are administered to a recipient after the cells have been contacted with said therapeutically effective amount of a PGE2 EP4 agonist or a pharmaceutically acceptable salt thereof
 12. The method according to claim 8, wherein the therapeutically effective amount of said PGE2 EP4 agonist or a pharmaceutically acceptable salt thereof is between about 0.001 μM to about 10 μM per about 1.0×106 per ml to about 1.0×107 per ml of said hematopoietic stem or progenitor cells.
 13. The method according to claim 8, wherein the hematopoietic stem or progenitor cells are harvested from marrow, umbilical cord or peripheral blood.
 14. The method according to claim 8, wherein the donor is a human.
 15. The method according to claim 8, wherein the donor is an animal.
 16. The method according to claim 11, wherein the donor and the recipient of the hematopoietic stem or progenitor cell are the same human or animal patient. 17.-24. (canceled) 